Normal 2 cell D Normal 4 cell E Polyspermy

Fig. 3.2.8 Fertilisation was scored by the absence (A) or presence of a fertilisation membrane (B), and normal cell cleavages (C and D). Polyspermy was also recorded and regarded as abnormal (E).

Individual female fecundity and gamete release were measured in 3 replicate 1ml samples using a Sedgewick Rafter counting cell and the number of eggs in 1L of seawater calculated. Checks were made to ensure optimum egg solutions were accurate (100-200 eggs ml-1). 100 eggs from each control were also counted and the occurrence of any fertilised eggs recorded. Any experiments where controls had a fertilisation rate >1% were discarded and, where possible repeated.

Individual male gamete release and concentrated sperm counts were accomplished using two methods. A haemocytometer counting cell was used to quantify the amount of sperm per ml-1 in three replicate counts of the 2003 samples of both starfish species. The 2004 samples of both species were quantified using a Coulter Multisizer II. Three replicate 500μl samples of each solution were analysed and the number of sperm quantified on a 1.9-20.0μm scale for particle size. Solutions were continually agitated to ensure thorough mixing using the in-built stirrer apparatus and the 500μl subsamples were counted within a 100μm tube. Serial dilutions of each sample had to be prepared using a standard control solution (isoton) to ensure accurate counts. A control (isoton) count was also undertaken in triplicate and recorded as a background count, which was subtracted from all other sperm counts. The concentration of sperm ml-1 calculated by both methods was converted into the number of sperm in 1L seawater. A 1ml subsample of dry sperm from both Marthasterias glacialis and Odontaster validus was quantified and compared

between the two methods to ensure that similar concentrations of sperm were being detected. This test was undertaken for a single male of each species in 2003 using the haemocytometer and in 2004 using the Coulter Multisizer II, and 3 replicate counts of each dry sperm sample were tested (Table. 3.2.2).

Table. 3.2.2 Dry sperm concentrations (ml-1) from individual males in 2003, using the haemocytometer counting cell, and in 2004 using the Coulter Multisizer II. Data are presented as overall means and +SD from 3 replicate counts.

Haemocytometer Mean SD Min Max

Marthasterias glacialis 2.10E+08 2.10E+07 1.92E+08 2.33E+08 Odontaster validus 3.18E+08 3.13E+07 2.98E+08 3.54E+08

Coulter Multisizer Mean SD Min Max

Marthasterias glacialis 1.70E+08 5.71E+06 1.65E+08 1.76E+08 Odontaster validus 3.88E+08 1.23E+07 3.74E+08 3.96E+08

Competence to spawn

Individual Odontaster validus were SCUBA diver collected from 15-20m in South Cove, adjacent to Rothera Research Station during the summer and winter field season of 2003-2004 (December 2003-August 2004). Individuals were maintained in the Bonner Laboratory’s flow-through aquarium facility. Monthly collections of individuals (10-15 starfish) were separated into individual aquaria and held in a thermally controlled experimental room adjacent to the main aquarium. Individuals were weighed wet (+0.01g) and measured using vernier callipers (+0.01mm) (R and r). Injections of 1-methyladenine were administered to induce spawning. The spawning competence of each individual starfish was assessed as the ability to release eggs or sperm. The starfish often contorted their body shape, curled up and raised themselves onto the tips of each arm. This behaviour was similar to the spawning posture often exhibited by natural populations of starfish. A lack of spawning was also noted.

Sperm Swimming Speed

The poor spawning response of individual Odontaster validus necessitated a more reliable Antarctic invertebrate to be selected and used in the sperm swimming trials.

The infaunal bivalve Laternula elliptica and the nemertean Parborlasia corrugatus were chosen and could be reliably strip spawned.

Experimental trials were implemented in the facilities of Rothera Research Station and the British Antarctic Survey, Cambridge aquarium during the Antarctic summer field season of 2003-2004 and during summer 2004 in the UK respectively. The infaunal bivalve Laternula elliptica and the nemertean Parborlasia corrugatus were SCUBA diver collected from 15-20m in Hanger Cove to the north of Rothera Research Station. Individuals were maintained in the Bonner Laboratory aquarium facility. A collection of these animals was also transported to the BAS headquarters on the RRS Ernest Shackleton in a purpose built container aquarium. The effects of temperature and viscosity on sperm swimming speed were studied. Individuals were strip spawned and a single male was required for each experimental trial. A single incision was made along the shell skirt to cut the adductor muscle of the hermaphrodite bivalve Laternula elliptica. The two shell valves were prized apart to allow access to the gonad (Fig. 3.2.9). A scalpel was used to make a small incision along the posterior section of the gonad. Care was taken not to puncture the gonad in an anterior position, to avoid eggs being released. Concentrated sperm was siphoned from the gonad using a sterile Pasteur pipette and deposited into a 30ml vial. Animal wet weight (+0.01g), shell length, width and height were also measured using vernier callipers (+0.01mm) (Fig. 3.2.9). Individual Parborlasia corrugatus were also strip spawned, however separate sexes could not be distinguished by external morphology. Therefore, dissections had to be made to find a suitable male specimen and any female individuals were discarded. A long incision was made down the length of each nemertean at a position sufficiently posterior to the mouth (1/3 animal’s length) to avoid the foregut. Packets of sperm were visible running laterally down the length of each male within the epithelium of the body wall and could be pierced using a sterile Pasteur pipette (Fig. 3.2.9). Concentrated sperm was also found free in the body cavity. Dry sperm was siphoned from each nemertean using a sterile Pasteur pipette and deposited into a 30ml vial. Individuals were weighed wet (+0.01g) and their retracted length (+0.01 mm) was also recorded (Fig. 3.2.9).

A B